Méndez J, Blanco L, Esteban J A, Bernad A, Salas M
Centro de Biología Molecular (Consejo Superior de Investigaciones Cientificas-Universidad Autónoma de Madrid), Spain.
Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9579-83. doi: 10.1073/pnas.89.20.9579.
Bacteriophage phi 29 DNA replication is initiated when a molecule of dAMP is covalently linked to a free molecule of the terminal protein, in a reaction catalyzed by the viral DNA polymerase. We demonstrate that single-stranded DNA molecules are active templates for the protein-primed initiation reaction and can be replicated by phi 29 DNA polymerase. Using synthetic oligonucleotides, we carried out a mutational analysis of the phi 29 DNA right end to evaluate the effect of nucleotide changes at the replication origin and to determine the precise initiation site. The results indicate that (i) there are no strict sequence requirements for protein-primed initiation on single-stranded DNA; (ii) initiation of replication occurs opposite the second nucleotide at the 3' end of the template; (iii) a terminal repetition of at least two nucleotides is required to efficiently elongate the initiation complex; and (iv) all the nucleotides of the template, including the 3' terminal one, are replicated. A sliding-back model is proposed in which a special transition step from initiation to elongation can account for these results. The possible implication of this mechanism for the fidelity of the initiation reaction is discussed. Since all the terminal protein-containing genomes have some sequence reiteration at the DNA ends, this proposed sliding-back model could be extrapolable to other systems that use proteins as primers.
噬菌体φ29 DNA复制起始于dAMP分子与末端蛋白的游离分子共价连接，此反应由病毒DNA聚合酶催化。我们证明单链DNA分子是蛋白质引发起始反应的活性模板，并且可以被φ29 DNA聚合酶复制。使用合成寡核苷酸，我们对φ29 DNA右端进行了突变分析，以评估复制起点处核苷酸变化的影响并确定精确的起始位点。结果表明：（i）在单链DNA上蛋白质引发起始没有严格的序列要求；（ii）复制起始发生在模板3'端第二个核苷酸的对面；（iii）需要至少两个核苷酸的末端重复才能有效地延长起始复合物；（iv）模板的所有核苷酸，包括3'末端的核苷酸，都被复制。提出了一种回滑模型，其中从起始到延伸的特殊过渡步骤可以解释这些结果。讨论了这种机制对起始反应保真度的可能影响。由于所有含末端蛋白的基因组在DNA末端都有一些序列重复，因此这种提出的回滑模型可能适用于其他使用蛋白质作为引物的系统。
